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Project Progress

The goal of this project is to identify changes that occur in the proteomes of permafrost bacteria in response to low temperature, high osmolarity, and other conditions associated with the permafrost environment. One line of experimentation involves the development of a novel 2-dimensional liquid fractionation method as a replacement for two-dimensional gel electrophoresis. The method uses a pH column-based separation in the first dimension followed by separation of the proteins in each pH fraction using nonporous silica (NPS) reversed phase high-performance chromatography ( HPLC). Using this method, we have been able to detect about 500 proteins in Psychrobacter 273-4. A comparison of cells grown at 22 versus 14°C has revealed that over 40 proteins are differentially expressed at these two temperatures, some being essentially qualitative changes. The identity of these polypeptides have been determined by using maxtrix-assisted laser desorption/ionization-time-of-flight ( MALDI-TOF) mass spectrometry and matching the peptide mass maps against the deoxyribonucleic acid ( DNA) sequence database that we have generated for Psychrobacter 273-4 in our genome sequencing project. Nearly a third of the proteins are conserved hypothetical polypeptides with unknown function, with most of the others being involved in translation or metabolism. A comparison of these results with the transcriptome results, suggests that posttranslational regulation may play a major role in temperature-regulated gene expression in Psychrobacter 273-4.

Proteome analysis has also been conduced with Psychrobacter cryopegella , a bacterium isolated from a briny water lens (-12 ° C) within the Siberian permafrost. P. cryopegella was found to grow at temperatures between -10 and 28 ° C and to remain physiologically active at -20 ° C. Two-dimensional gel analysis followed by liquid chromatography-double mass spectrometry was used to identify cold acclimation proteins (CAPs); i.e., proteins that were uniquely or preferentially expressed at temperatures below 0 ° C. Of the total 756 proteins that were detected, 24 were classified as CAPs. Of these, 20 were identified as open reading frames ( ORF) within the genome of Psychrobacter 273-4. Three of the ORFs were annotated as conserved hypothetical proteins. Of the remaining ORFs, 11 had previously been identified as stress response proteins in other organisms; these included superoxide dismutase, the ribosomal ribonucleic acid ( rRNA)-binding general stress protein Ctc, aspartate-semialdehyde dehydrogenase, the periplasmic component of a TRAP-type transport system, and the protein chaperone ppiB. The identities of these CAPs suggested the importance of oxidative stress, increased energy needs, and compatible solutes for growth at subzero temperatures.